1. Field of the Invention
The present invention relates to a method for preparing picornavirus virus-like particles (VLP) and recombinant baculoviruses used therein, and more particularly to a method for preparing enterovirus virus-like particles and recombinant baculoviruses used therein.
2. Description of the Prior Art
Enteroviruses belong to the Picornaviridae family and are positive-single stranded RNA viruses. Infection often happens via respiratory droplet or stool of an infected person, which can infect multiple systems and organs of a human body and even can cause organ failure. By serum neutralization test, enteroviruses can be classified into Poliovirus (PV), Coxsackie A virus (CAV), Coxsackie B virus (CBV), Enteric Cytopathogenic Human Orphan virus (i.e. Echoviruses), enteroviruses 68-71 and so on.
Being one of the more than 100 enteroviruses serotypes, enterovirus 71 (EV71) is the major etiological agent responsible for hand-foot-and-mouth disease (HFMD) in young children and infants. Children under 5 years of age are particularly susceptible to severe forms of EV71-associated neurological complications such as aseptic meningitis, brain stem encephalitis and even death. Since the 1998 outbreak in Taiwan that caused 405 severe cases and 78 deaths (Ho et al., 1999), in recent years the Asia-Pacific region (e.g. Taiwan, China, Malaysia, Japan and Vietnam) has experienced more frequent EV71-associated HFMD epidemics with high incidence of neurotropic complications and fatality rates (Tee et al., 2010). In 2008, the HFMD outbreak in Taiwan resulted in 373 severe cases and 14 deaths, all due to EV71 infection. The 2008 outbreak in China also led to 489097 reported cases that included 1125 severe cases, and claimed 126 lives. The increasing frequency of EV71 epidemics and high fatality rates underscore the urgent need to develop the vaccines.
VP1 is the major antigen and is highly variable among enteroviruses. Based on the sequences of VP1 gene, EV71 is divided into genogroups A, B and C and subgenogroups within genogroups B and C (B1-B5 and C1-C5).
Traditional viral vaccines include inactivated vaccines and attenuated vaccines, both containing the viral genetic materials and hence posing potential risks. Virus-like particles (VLPs) are empty particles consisting of viral structural proteins but devoid of viral nucleic acids, hence they are non-infectious. VLPs can generally induce broad and strong immune responses thanks to the preservation of many essential epitopes. Therefore VLPs have captured increasing attention as potential vaccine candidates.
Briefly, for the purpose of preventing enteroviruses infection, VLP is a promising vaccine candidate. Enterovirus possesses a positive single-stranded RNA genome that consists of a single open reading frame (ORF). The ORF expresses a large polyprotein that can be cleaved into P1, P2 and P3 regions (Brown and Pallansch, 1995). P1 region encodes the four structural proteins VP1, VP2, VP3 and VP4, while P2 and P3 regions encode other nonstructural proteins (e.g. 2A and 3CD) responsible for virus replication and virulence (McMinn, 2002). Based on a model derived from poliovirus, protease 2A autocatalytically cleaves P1 at its N-terminus and liberates P1 from the nascent polyprotein (Toyoda et al., 1986), while protease 3CD cleaves P1 precursor into VP1, VP3 and VP0 in trans. These three structural proteins spontaneously assemble into icosahedral procapsid in an ordered manner and proceeds through a series of intermediates, followed by the encapsidation of the RNA genome into the provirion. The final encapsidation step involves the cleavage of VP0 into VP2 and VP4, therefore the final mature virion consists of 60 copies each of VP1 and VP3, 58-59 copies of VP2 and VP4 and 2-1 copies of VP0.
Based on the knowledge, the baculovirus expression system has been previously used to co-express the P1 and 3CD proteins of EV71 in insect cells (Hu et al., 2003) and the formation of EV71 VLP in the infected insect cells was demonstrated. The recombinant baculovirus, Bac-P1-3CD, harboring the P1 gene under the polyhedrin promoter and the 3CD gene under the p10 promoter, is constructed using the traditional Bac-to-Bac system. Immunization of mice with the EV71 VLP triggered potent humoral and cellular immune responses and protected mice against lethal virus infection (Chung et al., 2008). However, the VLP yield was too low (≈10-20 μg/ml), making less attractive for commercial production.